Regulation of K+ in cellular and extracellular compartments is of central importance to volume regulation, fluid transport, the resting membrane potential and electrical activity in excitable cells. In heart, extracellular [K+] in narrow clefts between cells and in T-tubules is not always in diffusional equilibrium with external K+ ([K+]o ) and can be depleted or accumulated and thereby differ from [K+]o during a wide range of physiological/pathological conditions. The central goal of the project is to elucidate the consequences of extracellular K+ accumulation ([K+]a) on cardiac electrical activity in physiological and pathological conditions. To do so, we developed new K+ sensitive fluorescent probes that can be anchored to plasma membranes and upon binding to K+ produce a fluorescence change that reports the local [K+]. We can calibrate the probe, measure rapid (1 ms) changes in [K+]a in ventricular myocytes and Langendorff hearts. The Specific Aims are: Aim 1: Improve our new K+ sensitive probes consisting of: i) hydrophobic groups to anchor the probe to lipid bilayers, ii) hydrophilic groups to dissolve and prevent the probe from diffusing across the membrane into cells, iii) a fluorescent dye that senses iv) the binding of K+ to a K+- selective organic crown ether ring and exhibits a fluorescence change as a function of [K+] in the range of 1-50 mM. Aim 2: To test the hypothesis that different K+ currents contribute differently to [K+]a under different physiological conditions( ?? heartrate, ?? 2-adrenergic activity) and metabolic states. [K+]a responses will be compared during pharmacological interventions of channels responsible for K+ efflux (IK1, It,o, IK,slow, IKr, IKs, IKATP) and during manipulations of K+ re-uptake via the Na/K pumps. [K+]a will be compared in atrial and ventricular myocytes to test the effects of differences in APDs, K+ currents and T-tubules. In perfused hearts, APs and [K+]a will be simultaneously mapped, and changes in [K+]a will be measured on a beat-to-beat basis. Aim 3: To test the hypothesis that in pathological conditions, the rise of [K+]a can lead to regions of myocardium with an increase in excitability and a greater propensity to focal activity or premature beats that initiate arrhythmias. Simultaneous optical mapping of APs and [K+]a in perfused hearts will be used to measure APs and beat-to- beat changes in [K+]a transients to elucidate the role of [K+]a during arrhythmias elicited by ischemia/reperfusion applied either globally (zero flow) or locally via an LAD ligation. [K+]a elevation will be correlated with coronary vessels, fiber orientation and the origins of premature impulses. Such a project will develop K+ sensitive dyes for applications to cardiac electrophysiology and for the first time characterize [K+]a on a beat-to-beat basis. Dual optical mapping of [K+]a and APs will determine the changes in [K+]a that occur during physiological and pathological interventions, will validate methods to reduce [K+]a to offer new targets to treat cardiac diseases.